Project Summary/Abstract The bacterial chemotaxis system is the best-studied example of two-component signaling systems, a signal transduction motif that is widespread in prokaryotes and also found in some eukaryotes. The absence of two-component systems in mammals, and their importance for regulation of medically relevant phenomena such as biofilm formation of pathogenic microbes, make them potential targets for novel antibiotics. Additional important features in the chemotaxis system include the formation of large (~ 200 nm) membrane-bound hexagonal arrays of receptors that integrate signals to regulate the central kinase CheA. Successful approaches for interrogating receptor structure and mechanism within the these arrays it forms with CheA and CheW will be applicable to other large multi-protein machines that operate in cells. It is widely accepted that attractant binding to receptors causes a subtle conformational change in the periplasmic and transmembrane domains, but it is less clear how this exerts its inverse effects on activities of the cytoplasmic domain: inhibition of the CheA kinase and stimulation of receptor methylation. This project will investigate signal propagation through the cytoplasmic domain to CheA using native-like arrays of the E. coli Asp receptor cytoplasmic fragment (CF), CheA, and CheW assembled on vesicle templates or with molecular crowding agents. Biochemical assays and electron cryotomography are critical to the preparation of these homogeneous, native-like arrays in defined signaling states. Solid-state NMR distance measurements will measure CF dimer-dimer distances to measure the structure of the trimer of dimers and the proposed expansion of the membrane-proximal region during signaling. A novel combination of vesicle assembly of functional complexes with hydrogen exchange mass spectrometry will test proposals that regulated subdomain dynamics propagate the signal down to the tip of the receptor, and that changes in CheA domain dynamics limit or enhance productive interactions with the active site to control kinase activity. These studies will yield key insights into the molecular mechanism of transmembrane signaling by bacterial chemotaxis receptors to control the activity of the associated kinase CheA, and will also develop and demonstrate approaches for investigating how proteins operate in the large complexes that they form in the cell.